Photinus pyralis - Luciferase and its many tools
Introduction Luciferase is defined by a set of oxidative enzymes used in bioluminescence biologically distinct from light emitting photoproteins (1) . Firefly luciferin, initially a reaction observed as a light flashing in the tail of a common firefly, Photinus pyralis, was first isolated and purified in 1949 at Johns Hopkins University (2). Later, its synthesis and structure were determined by Dr. Emil H White, also at Johns Hopkins (2). The discovery of the luciferin-luciferase reaction opened many doors for molecular and cell biologists alike. This reaction is a robust detector of ATP production and its reaction yields light emission based on the amount of ATP present in the system. Uses in Molecular and Cell Biology Via genetic engineering, luciferase constructs can be produced for a variety of purposes. The luciferase gene may be synthesized and inserted into transfected cells, bacteria or another organism of choice. The luciferase reporter is generally used in molecular biology to asses the transcription of a gene region that has been altered to also transcribe the luciferase construct that's been added to the gene region of interest. Luciferase reporters have also been used in whole animal imaging. Differing types of cells can be constructed to produce luciferase, and thus can be traced as they travel about the vasculature of the animal. Luciferase has been reported in the examination of p53, ATP, multiple kinases, cytochrome P450 activity, caspase activity and many others. The detection of the luciferin-luciferase reaction is monitored via a luminometer which can measure the intensity of emitted light at yellow-green wavelength(1) (2) (3) (4) . Turning the green-yellow firefly.....red? Classically, light emission from the north american firefly is greenish yellow, around 550nm in wavelength. It is one of the most robust bioluminescence reactions known to science and time and time again has proven to be an incredibly useful tool to researchers worldwide. Branchini et al., sought to expand the use of luciferase into a multiple analyte assay that utilized a single substrate, luciferase (5) . In 1991, a team of Japanese researchers were able to mutate a single amino acid on five different residues, and had 5 different emissions of light, each at different wavelengths (6). With this in mind, Branchini et al., performed site directed mutagenesis to alter the active site of the enzyme. Two different mutants were synthesized; Val241Ile/Gly246Ala/Phe250Ser and Ser284Thr (5). Each of these mutations provided emission at different wavelenghts of light; WT at 557nm, triple mutant at 548nm and single mutant at 615nm (5). Equip with good signal separation and acceptable specific activity, researchers can utilize these P. pyralis mutant enzymes in dual color monitoring of genetic and/or dual analyte environmental screening methods, all while using a single substrate. Although, Branchini et al., had reported that these constructs are pH dependent (ph8) and are not promised to properly function at a wide range of pHs. Wang et al., recently proved this hypothesis correct (7). Even with the emission spectra not being as robust as its WT parent, these constructs are still very useful as a research tool and multiple companies in recent years have caught on, providing consumers with this product as it continues to improve scientific research. References #http://en.wikipedia.org/wiki/Luciferase #http://en.wikipedia.org/wiki/Firefly_luciferin #http://en.wikipedia.org/wiki/Photinus_pyralis #Gould SJ & Subramani S (1988) Firefly luciferase as a tool in molecular and cell biology. Analytical Biochemistry 175(1):5-13. #Branchini BR, Southworth TL, Khattak NF, Michelini E, & Roda A (2005) Red- and green-emitting firefly luciferase mutants for bioluminescent reporter applications. Anal Biochem 345(1):140-148 #Kajiyama N & Nakano E (1991) Isolation and characterization of mutants of firefly luciferase which produce different colors of light. Protein Engineering 4(6):691-693 #Wang Y, Akiyama H, Terakado K, & Nakatsu T (2013) Impact of site-directed mutant luciferase on quantitative green and orange/red emission intensities in firefly bioluminescence. Sci Rep 3:2490.